Retinopathy is one of the most prevalent and feared complications of diabetes. Although hyperglycemia is considered as the major cause of its development, the exact mechanism remains unclear. The overall aim of this proposal is to elucidate new insight into the pathogenesis of diabetic retinopathy. During the previous funding period we have demonstrated an important and novel role for a small molecular weight G- protein, H-Ras, in the accelerated apoptosis of retinal capillary cells. H-Ras is reported to upregulate matrix metalloproteinases-9 (MMP-9), a member of the family of metalloproteinases that regulate major biological functions, including apoptosis, inflammation and matrix degradation. Our central hypothesis is that 'activation of H-Ras cascade activates MMP-9, accelerating apoptosis of retinal capillary cells, and this ultimately leads to the development of retinopathy in diabetes'. In support, our exciting preliminary data show that MMP-9 is activated in the retina in diabetes. Inhibitors of H-Ras function prevent glucose-induced activation of MMP-9 in retinal capillary cells, and specific inhibitors of MMP-9 ameliorate increases in proinflammatory mediators and apoptosis. Aim 1 will determine the mechanism by which MMP-9 is activated in retinal microvasculature in diabetes. Using isolated retinal capillary cells and animal models of diabetic retinopathy, we will test the hypothesis that MMP-9 is activated in diabetes via H-Ras mediated signaling cascade, Raf-1-MAPK-ERK-NF-kB. Aim 2 will investigate the mechanism by which activated MMP-9 results in the development of diabetic retinopathy;and the hypothesis predicts that the activation of MMP-9 (a) damages mitochondria provoking apoptosis of retinal microvascular cells, and (b) increases inflammation and permeability, resulting in the development of retinopathy. This hypothesis will be tested in both in vitro and in vivo systems by pharmacologically or genetically manipulating MMP-9 gene. Studies have shown that diabetic retinopathy continues to progress for sometime after good glycemic control is re-instituted, suggesting a metabolic memory phenomenon. In aim 3, using a rat model, we will test the hypothesis that 'due to the continued activation of H-Ras after reversal of hyperglycemia, MMP-9 remains activated with elevated inflammatory mediators and continued capillary cell apoptosis. This is supported by our preliminary data showing the failure of retinal H-Ras activation to halt after re-establishment of normal glucose in diabetic rats. Understanding the signal transduction mechanisms responsible for the pathogenesis of diabetic retinopathy by characterizing the role of MMP-9 in the retina capillary cell death will provide fresh insight into the novel molecular targets for future pharmacological interventions to inhibit this sight-threatening complication of diabetes. This should help design specific therapeutics (e.g. inhibitors of MMP-9 and antisense) for future pharmacological interventions which patients can use to supplement their best possible glycemic control to treat this sight-threatening complication of diabetes. PUBLIC HEALTH RELEVANCE: This proposal is aimed at understanding the putative regulatory role(s) of matrix metalloproteinases in glucose-induced alterations in metabolic functions of retinal capillary cells leading to their demise, and the development of diabetic retinopathy. The results are expected to elucidate new insight into the pathogenesis of diabetic retinopathy, and reveal novel targets for therapies to prevent the development and progression of retinopathy.